Cholinergic Ligand–dependent Modulation of Oxidative Phosphorylation Coupling in Digitonin-permeabilized BE(2)-C Neuroblastoma Cells

Summary

We present a protocol to selectively permeabilize plasma membrane, enabling targeted delivery of substrates and inhibitors to assess mitochondrial oxidative phosphorylation (OXPHOS) in cultured cells, overcoming plasma membrane transport barriers that typically hinder such analysis. Using this technique, we demonstrated how OXPHOS function is modulated by muscarinic agonists and antagonists.

Abstract

The objective of this study was to assess oxidative phosphorylation (OXPHOS) function in cultured cells using defined substrate–inhibitor combinations while retaining cellular structure and cytosolic context lost in isolated mitochondrial preparations. Because intact cells are poorly permeable to several Krebs cycle intermediates, direct assessment of substrate-supported respiration through specific electron transport chain (ETC) entry points is limited. To overcome this, we applied digitonin-mediated selective plasma membrane permeabilization and performed extracellular flux analyzer–based coupling and electron flow assays in BE(2)-C neuroblastoma cells. To determine cell-type dependence, digitonin was empirically titrated in HEK293 cells and primary rat dorsal root ganglion (DRG) neurons using succinate + rotenone to isolate Complex II–IV–driven respiration.

Succinate-supported respiration with Complex I inhibition showed increased Complex II–IV–driven oxygen (O₂) consumption in permeabilized compared with non-permeabilized cells, consistent with improved access of a membrane-impermeant substrate to mitochondria. In contrast, respiration supported by substrates that enter via endogenous transport pathways (e.g., pyruvate/malate) showed smaller differences between conditions. Using this platform to test muscarinic ligands, we observed agonist- versus antagonist-associated differences in O₂ consumption in the coupling assay, whereas the electron flow assay revealed minimal ligand-associated effects under the tested conditions. These findings indicate that detectable ligand effects were more prominent at the level of coupling-defined respiratory states than maximal electron transfer capacity. Overall, selective permeabilization expands substrate accessibility in cultured-cell bioenergetic assays and enables analysis of pharmacologic modulation of mitochondrial respiration.

Introduction

Malfunction or reprogramming of mitochondrial energy metabolism is central to a wide spectrum of disorders, including neurodegeneration, cancer, inflammatory diseases, cardiometabolic conditions, and various genetic disorders. To better understand the role of mitochondrial metabolism in disease pathogenesis, several analytical strategies have been developed. These include measurements of O₂ consumption rate (OCR), extracellular acidification rate (ECAR), ATP quantification, substrate utilization, profiling or Krebs cycle metabolite analysis, and isotope tracing¹. Among these techniqu....

Protocol

1. Thawing and initial plating

NOTE: The base medium for BE(2)-C cells is a 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium and F12 Medium, supplemented with 10% fetal bovine serum (FBS).

1. Thaw frozen BE(2)-C cells from a cryovial in a 37 °C water bath. Once thawed, centrifuge the cells 200 × g for 5 min to remove the cryoprotectant.
2. Resuspend the cell pellet in complete growth medium and plate the cells in a 10 cm cell culture dish. Incubate the culture at 37 °C in a humidified incubator with 5% CO₂ in air until the cells reach sub-confluency.

Results

Optimization of digitonin concentration for BE(2) C Cell permeabilization in coupling assay
At the beginning of this protocol, we standardized the optimal digitonin concentration required for effective cell permeabilization without compromising mitochondrial respiration. In this protocol, we tested 5 µM and 10 µM digitonin as starting concentrations and performed a coupling assay. This assay evaluates the degree of coupling between the ETC and the OXPHOS machinery, allowing us to distinguish between .......

Discussion

This study introduces a rapid and cost-effective protocol for evaluating muscarinic ligand-mediated modulation of OXPHOS in cultured neuroblastoma cells. By employing the selective plasma membrane permeabilizer digitonin, in combination with defined substrate-inhibitor pairs, we enabled targeted delivery of metabolic substrates such as succinate directly to mitochondria in situ. This approach overcomes the limitations of intact cell membranes, which typically restrict access to key TCA cycle intermediates. Impor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetylcholine chloride	MilliporeSigma	A6625	Muscarinic agonist
ADP	MP Biomedicals, Inc	ICN19014901	Electron acceptor, substrate for ATP synthesis.
Antimycin A	AAJ63522MA	Thermo Scientific	Inhibits mitochondrial complex III
Atropine	MilliporeSigma	1044990	Muscarinic antagonist
Be2C cells	American Type Culture Collection	CRL-2268	A neuroblast cell that was isolated from the brain of a male patient with neuroblastoma, express muscarinic receptor
Digitonin	MilliporeSigma	D141	Mild detergent, membrane permeabilizer.
D-Mannitol	Thermo Scientific Chemicals	AA3334236	A non-metabolizable sugar alcohol, used for osmotic stabilization and membrane protection
EGTA	MilliporeSigma	E0396	Calcium Chelation, Prevents activation of calcium-dependent proteases and other enzymes that could degrade mitochondrial proteins.
fatty acid-free BSA	MilliporeSigma	126575	Prevents uncontrolled fatty acid effects, stabilizes mitochondria
FCCP	Cayman Chemical	NC0904863	A protonophore and uncoupler of oxidative phosphorylation in mitochondria
HEPES	MilliporeSigma	391340	pH buffering, compatibility with mitochondrial function
L-Ascorbate	TCI America	A053925G	TCA cycle intermediate, substrate of OXPHOS
Magnesium chloride	MilliporeSigma	M8266	For ionic balance, support for enzymatic activity
N1,N1,N1,N1-tetramethyl-1,4-phenylene diamine (TMPD)			Electron Donor to Cytochrome c, assessment of complex IV function by bypassing Complex I and II, TMPD/ascorbate enables direct measurement of Complex IV-dependent oxygen consumption
Oligomycin	MilliporeSigma	1404-19-9	Inhibits mitochondrial ATP synthesis, Complex V
Pirenzepine dihydrochloride	MilliporeSigma	P7412	Muscarinic biased agonist
Pyruvic acid	Thermo Scientific Chemicals	AC132155000	TCA cycle substrate
Rotenone	MilliporeSigma	557368	Inhibits mitochondrial complex I
Seahorse XF24 V7 PS Cell Culture Microplates	Agilent	102340-100	Cell Culture Microplates
Seahorse XFe24 Analyzer	Agilent	S7801BR	Agilent Seahorse XFe24 Analyzers measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format.
Succinic acid	Fisher Chemical	AC158742500	TCA cycle intermediate, substrate of OXPHOS
XFe24 sensor cartridge calibration buffer	Agilent	102340100	PBS buffer pH 7.2
XFe24 sensor cartridges	Agilent	102340-100	sensor for OCR and ECAR measurement